Selection of monoclonal antibodies which bind specific antigens presents a major analytical challenge when the antigen source is impure. The gel electrophoresis procedure is often used in analyzing the antigens present. In this procedure, a gel, commonly polyacrylamide, is poured into a rectangular plate and the protein or antigen mix to be analyzed is applied to the top of the gel. A strong voltage is then applied between the top and bottom of the gel to pull the charged proteins through the gel which, after a period of time, results in a separation of the proteins present into separate bands spaced between the top and bottom of the gel. A carrier sheet, typically a nitrocellulose membrane, is then brought into contact with the surface of the gel causing the proteins to adhere to and become immobilized on the surface of the nitrocellulose carrier sheet. In the present typical immunoblotting technique, the sheet of nitrocellulose with the proteins adhering thereto is cut up into several strips and each strip is incubated for a period of time in a solution containing a particular antibody of a first type, designated primary antibody. If the primary antibody recognizes one of the bands of protein on the strip, it will bind to it. The strip may then be placed in a solution containing a secondary antibody which recognizes and binds to the primary antibody, with the secondary antibody having a tracer (e.g., a radioactive isotope) carried therewith. If a radioactive isotope tracer is used, a film sensitive to the radiation from the isotope may be laid over the strip to reveal the location on the strip at which the antibodies have bound. In this way, the proteins to which the particular antibodies are binding can be determined.
The standard immunoblotting technique briefly described above can thus be very time consuming and tedious, particularly if tens or hundreds of antibodies are to be tested. The technique is also prone to errors, since the separate strips may become mixed up or misidentified, and the strips themselves can tend to shrink or expand, making it difficult to compare the position of the bands detected on separate strips. In particular, selection of hybridoma cultures producing monoclonal antibodies makes efficient and rapid screening of large numbers of cultures highly desirable. Usually, immunoblotting is the only method by which the monoclonal antibodies may be selected if the antigen of interest is not purified. In the early stages of the selection of hybridomas, colonies must be screened within days to ensure cell viability. If the screening method is inefficient at this stage, extended cell culture work is required. More efficient screening techniques permit the screening of more hybridomas, increasing the probability of success in selecting the desirable antibody producing lines. However, present immunoblotting screening is inefficient for several reasons. First, for each hybridoma to be tested, a separate strip of nitrocellulose must be processed through the many steps of the procedure. Second, during the early stages of hybridoma growth, the quantity of antibody available for screening is extremely limited, and individual strips of nitrocellulose must be incubated with the limited quantity of antibody, a difficult proposition. Third, all of the individual strips of nitrocellulose must be compared after completion of the procedure, which is time consuming and is subject to the errors described above.